Preparations of routine diagnostic media, culture and isolation of bacteria

1. The University of Zambia
School of veterinary medicine
Department of paraclinical studies
Name: Musalo Brian
Computer #: 10008047
Course code: VMP-4300 (Immunology & Bacteriology)
Lab: Practical One-Preparations of routine diagnostic media, culture and isolation of
bacteria
Attention: Mr.mubita
Date:19/11/13
Copyright: Musalo Brian Chisanga|Bacteriology/Immunology Lab Report|2013|

2. Title:preparation of routine diagnostic media, culture and Isolation of Bacteria
Aim:The purpose of this exercise is for the student to experience the process of
Preparing bacterial media.
Introduction:
A growth medium or culture medium is a liquid or gel designed to support the growth of
microorganisms or cells,or small plants like the mosspatens. There are different types of media
for growing different types of cells. There are two major types of growth media: those used for
cell culture, which use specific cell types derived from plants or animals, and microbiological
culture, which are used for growing microorganisms, such as bacteria. The most common growth
media for microorganisms are nutrient broths and agar plates; specialized media are sometimes
required for microorganism and cell culture growth.
When Cultivating Bacteria In 1861 Pasteur prepared fluid culture media which later led to the
development of improved bacteriological media. In 1875 Robert Koch succeeded in making
solid media which helped to isolate bacteria in pure culture. During the last quarters of the 19th
century a succession of discoveries was reported, bearing on the relation of bacteria to human
and human disease, which opened a new era in medicine. These included Pasteur’s isolation of
the bacillus of fowl cholera, Koch’s discovering of tubercle bacillus, Loffler’s discovery of
diphtheria bacillus and the bacillus of swine erysipelas, Kitts discovery of bacillus of
hemorrhagicsepticemia of cattle, and isolation of Clostridium tetani the causative agent of
tetanus. These individuals provided landmarks to preparing diagnostic media in their studies and
discoveries.
The use of a basal media is the backbone of all media (Nester W.E et al, 2004). In
this experiment two types of media are used; selective and differential media. Selective media
are employed for selected /specific microorganisms for example MacConkey agar is a selective
media for Escherichiacoli and salmonella. It is used to isolate gram negative because of its
characteristic crystal violet color that inhibit gram positive bacteria and bile salts also inhibit
most of the gastrointestinal bacteria: These collectively perform the role of differential media
such that they are able to distinguish lactose fermenting and non-lactose fermenting bacteria,
Escherichiacoli and salmonella respectively. An example of a differential media is Blood agar
base, this can be made by addition of 10% defibrinated sheep blood to basal agar. Besides being
nutritious, blood agar has differential properties and is used to detect bacteria. Blood agar base
and MacConkey agar are used in this experiment, they come in the form of powder and to
convert them into a solid form you need to rehydrate them. They both contain agar which is an
extract of seaweed that has no nutritional value to bacteria. Agar is added to the medium for the
sole purpose of producing a solid surface on which the bacteria can grow. The addition of agar to
the media has other desirable attributes. It remains in a solid state even at high temperatures and
does not liquefy until it reaches 100°C. This allows the growth of bacteria that are thermopiles
Copyright: Musalo Brian Chisanga|Bacteriology/Immunology Lab Report|2013|

3. (like temperatures > 40°C) within the laboratories. Agar solidifies at approximately 40° C. Also
very few bacteria have to ability to decompose the agar so it remains stable even while bacteria
grow upon or within it. Buffers are put into the culture medium to keep the pH of the medium
from changing drastically. Most bacteria prefer a pH around neutral. As the bacteria grow and
reproduce on the medium, acidic and basic by-products of metabolism are produced. If buffers
were not included in the medium, the bacteria would be quickly killed off by their own waste
products.
Materials:
Commercial powder(media)-dehydrated media
Conical flask/ Elein Meyer flask
Spatula
Distilled water
Electronic weighing balance
Measuring cylinder
Bunsen burner
Microwave
Bacteriology loops
Autoclave
Petri dish
70% alcohol
Fume hood
Aluminum foil
Piece of paper
Samples; intestinal washing & milk sample suspected of mastitis
Copyright: Musalo Brian Chisanga|Bacteriology/Immunology Lab Report|2013|

4. Procedures:
A. Preparation of MacConkey agar
Commercial dehydrated powder was collected using a spatula and was weighed on an
electronic weighing balance. Since 44.5g of MacConkey should be added to 1L (1000ml)
of distilled water, for the needed 200ml, 8.9g of MacConkey powder was added. The
powder was added using a piece of paper into a conical flask containing a bit distilled
water from the total measured 200ml so as to avoid the media powder sticking at the
bottom of the flask. Then afterwards the rest of the distilled water was added to make a
total of 200ml. the flask was then put into the microwave carefully for 5 minutes to
ensure that the media powder thoroughly gets dissolved. Afterwards the conical flask top
was covered with an aluminum foil and the flask was autoclaved for 15 minutes at
121degrees Celsius. Afterwards then flak was left to cool to about 45-50 degrees Celsius.
Afterwards the media was prepared aseptically into the petri dishes using a safety hood
with a Bunsen burner on after disinfection with 70% alcohol. Then the media was left to
cool until it was needed for use.
B. Preparation of blood agar
8.5g of media powder was dissolved in 200ml of distilled water (21.25g in 500ml) in a
conical flask. This was then put in a microwave for about 5 minutes to dissolve the media
powder. Then the mouth of the conical flask was covered with aluminum foil and was
autoclaved for 15 minutes at 121 degrees Celsius. Afterwards 10% sheep blood (20ml of
blood in 200mlof distilled water) was aseptically poured into a conical flask and mixed
thoroughly. Then afterwards the mixture was poured aseptically into the petri dishes. The
bubbles created on the surface of the solution were destroyed using a blue Bunsen flame.
Then the plates were left to cool for future use.
C. Culture and isolation of bacteria
2 samples were provided in the experiment: milk sample suspected of mastitis &
intestinal washing. A wire loop was used to transfer samples from a test tube to the
media. The wire loop were sterilized by heating them over a Bunsen burner blue flame. A
loop full of the sample was transferred from the test tube to the media and streak plate
method was used i.e. loop was streaked on the plate. 4 zones were created namely zones
A, B, C and D then the growth of the cultured samples were observed and results were
obtained. The plates were then put in an incubator at 37 degrees Celsius.
Copyright: Musalo Brian Chisanga|Bacteriology/Immunology Lab Report|2013|

5. Results & Discussion:
In this experiment blood agar and MacConkey were used. These were prepared through
the common dilution of commercial powders method. The dilutions were then thoroughly
autoclaved at 121 degrees Celsius in order to ensure that the samples and apparatus being
used are sterilized to avoid foreign bodies from interfering with the experiment. The
autoclave operates on the principal of the pressure cooker. The plugged flask should be
placed in the autoclave and it must be ensured that enough water is poured beneath the
rack that it just comes up to the lip inside the chamber. The lid must be closed by hooking
the claw over the copper bar with a snap. Press "reset", make certain that the exhaust is
set to "Slow -- liquids" and set the timer for 15 minutes. When the autoclave is done (i.e.,
light is out, pressure is at "0" instead of at 15 psi and temp is < 121°C), open the door
carefully while wearing autoclave gloves. Open the door from the side so you do not get
"blasted" by hot steam. Once the initial release of steam is done, open the door the rest of
the way. Remove your flask, while wearing autoclave gloves, of the melted and sterile
agar. Place your flask of HOT agar in an open place. This will allow the agar to cool, but
not to go less than 40°C when it will solidify). Let it set for about 30 minutes. Once the
thirty minutes have passed, remove the warm agar from the open, remove the plug from
the opening of the flask and begin pouring the mixture into the small portion of the petri
dish. You need only pour about 0.5 - 0.75 cm of agar into the plate. Cover the plate
before advancing to the next plate. Repeat the procedure until all plates have melted agar
in them. The pouring of agar was aseptically done and this should be noted. Dispose of
the left-over agar and rinse your flask as directed by your instructor. Let the agar plates
cool until solid, then turn them upside down for storage in the refrigerator. These plates
can be used throughout the semester/term. After all this was done, the samples under test
were inoculated on the prepared media i.e. milk sample suspected of mastitis and
intestinal washing were both streaked onto the plates containing blood agar &
MacConkey and these were incubated at 37 degrees Celsius to provide a life supporting
ecology for bacterial growth
Conclusion:
Blood agar and MacConkey agar mediawere prepared by means of diluting their
commercial powders and their colors were red and red wine respectively. The plates were
then put in an incubator at the temperature of 37 degrees Celsius with streaks of milk
sample suspected of mastitis and intestinal washing overnight. The prepared media was
very viable as bacteria cultures were seen on the plates the following day
Copyright: Musalo Brian Chisanga|Bacteriology/Immunology Lab Report|2013|

6. References:
Blood D.C& Studdert P.V, 1988,Bailliere tinda comprehensive veterinary
dictionary, 1st edition,Baillieretinda, Saunders, pages 8,242.
Hawkey M.P and Lewis A.D, 1989, medical bacteriology and practical
approach, IRC Press at oxford university press, England, page8.
Nester W.E et al, 2004, microbiology: A human perspective, 4th edition, McGraw
Hill, New York, pages 83, 84, and 94.
DIFCO Manual: Dehydrated Culture Media and Reagents, 10th Edition. (Difco
Laboratories: Detroit) ©1984, Pp. 543, 546, 616, 1023.
Copyright: Musalo Brian Chisanga|Bacteriology/Immunology Lab Report|2013|